Tuesday, September 16, 2008

How RNA Polymerase Binds to DNA

Most cells contain two forms of RNA polymerase. The "core" polymerase is the part that carries out transcription of a gene where the DNA sequence is copied to produce a single-stranded RNA molecule. The core polymerase binds DNA non-specifically as you might expect for a DNA binding protein that has to travel down a large number of different genes.

When transcription is terminated the core RNA polymerase is released. In order to start a new round of transcription, the core RNA polymerase has to be directed to bind at a promoter, defined as the specific DNA sequence where transcription is initiated. There are specific DNA binding factors that bind to promoters and to RNA polymerase. That's how they direct RNA polymerase to the place where transcription has to start. These factors bind first to core polymerase forming the second form of RNA polymerase called the holoenzyme.

The binding parameters of the E. coli core polymerase and the holoenzyme have been studied in detail. In E. coli cells there are several different versions of holoenzyme. Each one contains a different initiation factor that binds to a different series of promoters. The most common initiation factor is called σ70 (sigma-70) and it binds to most of the promoters in the cell.

The steps in transcription initiation are shown in the figure. First, holoenzyme consisting of core polymerase + σ70, binds non-specifically to any stretch of DNA. It then moves along the DNA in a one-dimensional search until it finds a promoter sequence. This is followed by a local unwinding of the DNA and synthesis of a short piece of DNA.

Subsequent steps (not shown) require the dissociation of the initiation factor (σ70) and the formation of an elongation complex. RNA polymerase is then free to leave the promoter region and move down the gene making RNA.

THEME:TranscriptionThe same kinds of parameters that we discussed yesterday are used to describe RNA polymerase binding [see DNA Binding Proteins and Repression of the lac Operon]. The core RNA polymerase by itself binds DNA non-specifically with an association, or binding, constant (Ka) of 1010 M-1. This is very tight binding for a DNA binding protein. Once bound to DNA the core RNA polymerase dissociates very slowly (t1/2 = 60 minutes).

The holoenzyme can also bind non-specifically. In this case the association constant is 5 × 106 M-1 and the complex dissociates rapidly (t1/2 = 3 seconds). The holoenzyme binds specifically to promoter sequences with an association constant of 2 × 1011 M-1 and t1/2 = 2 to 3 hours. Thus, the interaction of the initiation factor with core RNA polymerase has two effects: it decreases the affinity for random stretches of DNA and increases the affinity for the promoter sequence.

A typical E. coli cell contains about 5000 molecules of RNA polymerase. When the cells are growing rapidly, 2500 molecules will be bound to genes in transcription complexes. Another 1250 will be in initiation complexes of various sorts and most of the remaining RNA polymerase molecules (1200) will be bound to DNA non-specifically. Only a small number (~50) will be free in the cytoplasm.

Since the holoenzyme molecules are capable of initiating transcription on their own, a small number of the non-specifically bound molecules will accidentally transcribe short stretches of DNA. These spurious transcripts don't usually cause a problem since they are quite rare. Nevertheless, their presence means that much of the intergenic DNA in the E. coli genome is transcribed at one time or another.

Eukaryotic cells contain three different kinds of RNA polymerases [Eukaryotic RNA Polymerases]. Each one is much more complex that the bacterial enzymes but the principles of transcription initiation are the same.

In eukaryotes there are about a dozen general initiation factors for each of the different RNA polymerases. The ones for RNA polymerase II—the enzyme that transcribes protein-encoding genes—are called transcription factor IID (TFIID) etc. All of the factors are required for specific RNA polymerase binding at a promoter site and all of them associate with the core RNA polymerase to form a large holoenzyme complex. The eukaryotic general initiation factors do the same thing for eukaryotic core polymerase as the bacterial ones do for the bacterial RNA polymerase; they convert the complex to a specific DNA binding protein and lower its affinity for binding non-specifically.

As is the case in bacteria, a substantial number of holoenzyme complexes will be bound non-specifically to DNA at any one time. The proportion is much, much higher in mammalian cells because of the presence of so much junk DNA in the genome. This has the effect of soaking up a lot of holoenzyme complexes.

Since the holenzyme complexes, like those in bacteria, are capable of initiating basal levels of transcription, we should not be surprised to find spurious transciption in all parts of the genome. These transcript will be rare but they will come from any site where RNA polymerase holoenzme can bind.

These transcripts will be rare but they will come from any site where RNA polymerase holoenzme can bind.

Exactly how rare is the key question. Since most of the human genome is repetitive, spurious transcription would predominantly produce RNA to these repetitive sequences. Presumably, there is no strand preference to the initiation of this spurious transcription, so (+) and (-) strands of repetitive regions will be produced with approximately equal frequency. These (+) and (-) repetitive sequence transcripts are complementary to each other and should be able to anneal to form double-stranded RNA to these repetitive regions. Is this dsRNA to human repetitive genomic elements detected?

Laurence A. Moran

Larry Moran is a Professor Emeritus in the Department of Biochemistry at the University of Toronto. You can contact him by looking up his email address on the University of Toronto website.

Sandwalk

The Sandwalk is the path behind the home of Charles Darwin where he used to walk every day, thinking about science. You can see the path in the woods in the upper left-hand corner of this image.

Disclaimer

Some readers of this blog may be under the impression that my personal opinions represent the official position of Canada, the Province of Ontario, the City of Toronto, the University of Toronto, the Faculty of Medicine, or the Department of Biochemistry. All of these institutions, plus every single one of my colleagues, students, friends, and relatives, want you to know that I do not speak for them. You should also know that they don't speak for me.

Subscribe to Sandwalk

Quotations

The old argument of design in nature, as given by Paley, which formerly seemed to me to be so conclusive, fails, now that the law of natural selection has been discovered. We can no longer argue that, for instance, the beautiful hinge of a bivalve shell must have been made by an intelligent being, like the hinge of a door by man. There seems to be no more design in the variability of organic beings and in the action of natural selection, than in the course which the wind blows.Charles Darwin (c1880)Although I am fully convinced of the truth of the views given in this volume, I by no means expect to convince experienced naturalists whose minds are stocked with a multitude of facts all viewed, during a long course of years, from a point of view directly opposite to mine. It is so easy to hide our ignorance under such expressions as "plan of creation," "unity of design," etc., and to think that we give an explanation when we only restate a fact. Any one whose disposition leads him to attach more weight to unexplained difficulties than to the explanation of a certain number of facts will certainly reject the theory.

Charles Darwin (1859)Science reveals where religion conceals. Where religion purports to explain, it actually resorts to tautology. To assert that "God did it" is no more than an admission of ignorance dressed deceitfully as an explanation...

Quotations

The world is not inhabited exclusively by fools, and when a subject arouses intense interest, as this one has, something other than semantics is usually at stake.
Stephen Jay Gould (1982)
I have championed contingency, and will continue to do so, because its large realm and legitimate claims have been so poorly attended by evolutionary scientists who cannot discern the beat of this different drummer while their brains and ears remain tuned to only the sounds of general theory.
Stephen Jay Gould (2002) p.1339
The essence of Darwinism lies in its claim that natural selection creates the fit. Variation is ubiquitous and random in direction. It supplies raw material only. Natural selection directs the course of evolutionary change.
Stephen Jay Gould (1977)
Rudyard Kipling asked how the leopard got its spots, the rhino its wrinkled skin. He called his answers "just-so stories." When evolutionists try to explain form and behavior, they also tell just-so stories—and the agent is natural selection. Virtuosity in invention replaces testability as the criterion for acceptance.
Stephen Jay Gould (1980)
Since 'change of gene frequencies in populations' is the 'official' definition of evolution, randomness has transgressed Darwin's border and asserted itself as an agent of evolutionary change.
Stephen Jay Gould (1983) p.335
The first commandment for all versions of NOMA might be summarized by stating: "Thou shalt not mix the magisteria by claiming that God directly ordains important events in the history of nature by special interference knowable only through revelation and not accessible to science." In common parlance, we refer to such special interference as "miracle"—operationally defined as a unique and temporary suspension of natural law to reorder the facts of nature by divine fiat.
Stephen Jay Gould (1999) p.84

Quotations

My own view is that conclusions about the evolution of human behavior should be based on research at least as rigorous as that used in studying nonhuman animals. And if you read the animal behavior journals, you'll see that this requirement sets the bar pretty high, so that many assertions about evolutionary psychology sink without a trace.

Jerry Coyne
Why Evolution Is TrueI once made the remark that two things disappeared in 1990: one was communism, the other was biochemistry and that only one of them should be allowed to come back.

Sydney Brenner
TIBS Dec. 2000
It is naïve to think that if a species' environment changes the species must adapt or else become extinct.... Just as a changed environment need not set in motion selection for new adaptations, new adaptations may evolve in an unchanging environment if new mutations arise that are superior to any pre-existing variations

Douglas Futuyma
One of the most frightening things in the Western world, and in this country in particular, is the number of people who believe in things that are scientifically false. If someone tells me that the earth is less than 10,000 years old, in my opinion he should see a psychiatrist.

Francis Crick
There will be no difficulty in computers being adapted to biology. There will be luddites. But they will be buried.

Sydney Brenner
An atheist before Darwin could have said, following Hume: 'I have no explanation for complex biological design. All I know is that God isn't a good explanation, so we must wait and hope that somebody comes up with a better one.' I can't help feeling that such a position, though logically sound, would have left one feeling pretty unsatisfied, and that although atheism might have been logically tenable before Darwin, Darwin made it possible to be an intellectually fulfilled atheist

Richard Dawkins
Another curious aspect of the theory of evolution is that everybody thinks he understand it. I mean philosophers, social scientists, and so on. While in fact very few people understand it, actually as it stands, even as it stood when Darwin expressed it, and even less as we now may be able to understand it in biology.

Jacques Monod
The false view of evolution as a process of global optimizing has been applied literally by engineers who, taken in by a mistaken metaphor, have attempted to find globally optimal solutions to design problems by writing programs that model evolution by natural selection.